The Frequency of Resistance Genes in Salmonella enteritidis Strains Isolated from Cattle

Background: Salmonella enteritidis causes infections in humans and animals. Antibiotics are used to eliminate bacterial infections, which become resistant to antibiotics after a while. This study aimed to isolate S. enteritidis from cattle feces samples and also to evaluate the frequency of genes associated with multi-drug resistance (MDR). Methods: One hundred ten fecal samples of cattle were collected from Jul to Dec, 2017 in Khuzestan Province, southern Iran. Bacterial culture and molecular methods were used to isolate and identify S. enteritidis. Disk diffusion method was used to assess antibiotic susceptibility. Then Polymerase chain reaction (PCR), assay was used for definitive diagnosis of S. enteritidis and resistance genes. Results: Overall, 101 (91.81%) samples were detected to be contaminated with Salmonella genus and 86 samples (85.14%) were identified as S. enteritidis. The highest and lowest antibiotic resistance were belonged to gentamicin (n=70, 81.39%), and tetracycline (n=6, 6.97%). Besides, 64 samples (74.42%) had 2–10 drugs resistance patterns. Moreover, the highest and the least resistance were related to blaIMP-1 (n=73, 84.88%) and tet(B) (n=49, 56.97%) genes respectively. Conclusion: The drug-induced genes in S. enteritidis have a high frequency. Therefore, antibiotic resistance and high MDR to antibiotics can be due to the incorrect use of antibiotics and the lack of health monitoring in Cattle farms.


Introduction
Salmonella is one of the most important members of the Enterobacteriaceae family. This bacterium is a facultative inter-cellular, gram-negative, rodshaped, bacilli-shaped, spore-free, aerobic and anaerobic pathogen (1). Most of the pathogenic Salmonella species that cause human disease belong to the S. enterica species, which live on the host gastrointestinal tract. S. enteritidis (S. enteritidis) is among the most important disease-causing serotypes. So far, more than 2,500 Salmonella serovars have been identified based on flagellar and somatic antigens, most of which are pathogenic to humans and animals (2,3). Salmonella in humans causes diseases such as salmonellosis, intestinal or typhoid fever, septicemia and gastroenteritis. In this regard, S. enteritidis plays a significant role in the development of these infections (4,5). Diseases transmitted through food are the most important health issues. Salmonella is often found in food, meat and dairy products, related to the livestock industry (6,7). The use of antibiotics for humans and animals causes major health problems, including the presence of antibiotic residues in body tissues and livestock products and the resistance of pathogens to antibiotics, leading to the selection of pathogenic bacteria resistant to multiple drugs (4,8). Multidrug resistance (MDR) in bacteria is caused by various mechanisms, including reactive changes of the target protein and the enzymatic inactivation of the drug. Enzymatic inactivation is a common resistance mechanism for natural antibiotics such as aminoglycosides and β-lactams (9)(10)(11). Due to increasing rate of MDR bacterium, the purpose of this study was to use PCR as a precise method for detecting S. enteritidis and also to determine the frequency of resistance genes of this bacterium in cattle samples in Iran.

Sampling
The present study was conducted from Jul to Dec 2017 in Khuzestan Province, southern Iran. Overall, 110 cattle feces samples were collected randomly and used for detection of Salmonella.

Isolation and Culture of Salmonella
The feces samples were transferred to concentrated peptone water and incubated at 37 ℃ for 24 h. Then, the samples were transferred to Rappaport Vassiliadis (RV) and incubated again at 43 ℃ for 24 h. The isolated bacteria were cultured on Salmonella Shigella Agar (SSA). Black and gray colonies were considered as suspected Salmonella. Complementary assays and differential microbial tests (i.e., IMViC and urease) and culturing on Triple Sugar Iron Agar (TSI) according to Bergey's manual were used to detect Salmonella strain. The lysine decarboxylation was performed on isolated bacteria by culturing in lysine decarboxylase broth to identify the Salmonella family. Sulfide Indole Motility medium (SIM) was used for direct detection of Salmonella spp. by sulfide production, indole formation, and motility.

Gene Amplification
Genomic DNA was extracted using QIAamp mini kit (Qiagen, GmbH, Germany). PCR method using specific oligonucleotide primers for 16S rRNA gene was performed for the direct detection of Salmonella family. In the next step, Salmonella-encoded fimbria (sefA) gene was targeted for specific detection of S. enteritidis from other Salmonella strains. The primer sequences used in the present study are shown in Table 1. PCR reactions were performed in a total volume of 25 μL in 0.2 ml tubes containing 2 μL of template DNA, 1 μM of each primer, 2 mM MgCl2, 5 μL of 10X PCR buffer AMS, 200 μM dNTPs, and 1 unit of Taq DNA polymerase (CinnaClon Co, Iran). The PCR assay was performed at 95 ℃ for 5 min and then for 32 cycles of 94 ℃ for 1 min, annealing temp according to Table 1 for 40 sec, 72 ℃ for 40 sec, and a final extension at 72 ℃ for 5 min, with a final hold at 10 ℃ in a thermal cycler (Mastercycler gradient, Eppendorf, Germany). The PCR amplified products were detected in 2% EtBr stained agarose gel electrophoresis.

Antimicrobial drug susceptibility tests
The colonies were cultured on Nutrient Agar (Merck, Darmstadt, Germany) at 37 ℃ for 24 h after detection of positive S. enteritidis samples from other Salmonella strains. Then, the colonies were sub-cultured into Nutrient Broth (Merck, Germany) and incubated at 37 ℃ in a shaking incubator for 14 h. McFarland 0.5 turbidity standards were used for bacterial turbidity. After standardization, 100 µL of bacterial suspensions were cultured on Mueller-Hinton agar medium (Merck, Darmstadt, Germany). Drug resistance testing was performed using standard Bauer-Kirby disk diffusion method for all samples (positive culture) according to CLSI protocols for determination of antibiotic susceptibility patterns of S. enteritidis isolated from feces specimens and the cultures were incubated at 37 ℃ for 24 h. The antibiotic disks were used for determination of antibiotic susceptibility patterns of S. enteritidis isolated from feces specimens.

Detection of antibiotic resistance genes
The PCR was performed for identification of resistance genes. The primers were blasted at the NCBI using the experimental GENINFO BLAST Network Service to assess degree of homology between these primers and other reported sequences and at the end were obtained from CinnaClon Co, Iran. The specific oligonucleotide primers for gene amplification of antimicrobial resistance genes in isolated S. enteritidis and the sequences of primers are available in

Statistical Analysis
All statistical tests were performed using Graph Pad Prism statistical software, ver. 7.00 (Graph Pad, San Diego, CA, USA). For all tests, P-value <0.05 was considered statistically significant.

Identification of bacteria
Out of 110 samples, 101 (91.81%) samples were positive for Salmonella by detecting 258 bp band of 16S rRNA gene. Furthermore, the numbers of 86 (85.14%) specimen were sefA gene positive (488 bp).

Resistance genes
In the studied samples, the highest resistance belonged to the blaIMP-1 genes (n=73, 84.88%) and the lowest resistance, on the other hand, was for tet(B) (n=49, 56/97%) gene. Detection of resistance genes in isolated S. enteritidis using PCR technique revealed fragments with specific length sizes in Table 1.
The phenomenon of antimicrobial resistance has been an urgent global problem since the 1990s (21). The excessive and incorrect use of antimicrobial agents and lack of appropriate infection control are the main possible reasons for this alarming phenomenon and development of MDR in bacteria (22). In a study, the rate of 52% MDR was shown for Cattle (23 (24). However, in present study, the highest resistance phenotypes were streptomycin, gentamicin, sulfamethoxazole, amikacin, chloramphenicol and imipenem antibiotics. Due to the presence of streptomycin and sulfamethoxazole antibiotics and the creation of resistance phenotypes in both studies, care should be taken in their use.

Conclusion
The comparison of the results of present study with others suggests that both the prevalence and the antimicrobial resistance pattern for Salmonella is different based on geographical zones. This study determined the widely spread prevalence of S. enteritidis and MDR in cattle from Khuzestan Province. Efforts that include further implementation of hazard analysis of critical control in livestock production are needed to reduce the incidence of Salmonella.

Ethical considerations
Ethical issues (Including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc.) have been completely observed by the authors.